With the sequence of the mouse genome essentially complete, the new long-term goal is to assign function to the genes identified. Knowing the functions for the complete set of genes in a model mammalian organism has obvious biological significance. The value of this information for human health and medicine is in its potential to aid in the discovery of new disease-related genes, to provide the tools needed to create new models for human disease, and to help validate new drug targets. The Knock-Out Mouse Project (KOMP) has set the goal of creating a comprehensive resource of mouse mutations in which every gene in the mouse genome has been knocked out by a null mutation marked with a reporter system. To apply the power of targeted gene modification on the ambitious scale proposed by the KOMP demands significant technological improvements in throughput and capacity. We propose to use our high-throughput and validated VelociGene technology to create a library of 10,000 targeting vectors and a collection of 10,000 mouse ES cell clones, each carrying a definitive null mutation for a unique protein-coding gene. To meet our ambitious production targets, we describe a research plan organized to achieve the following specific aims: 1. Construct a library of BAC-based targeting vectors (BacVecs) designed to create definitive null mutations(in most cases, deletions of the entire coding region) and incorporating a lacZ reporter of high utility for 10,000 mouse protein-coding genes. 2. Using the BacVecs produced by Specific Aim 1, create a collection of C57BL/6 ES cell clones each having a defined null allele and an incorporated reporter for each of 10,000 mouse protein-coding genes,and generate mice from 5% of these ES cell clones (500 mouse lines total) as a test of the collection for germ-line transmission. 3. Create a library of BAC-containing recombination-ready E. coli clones (from Specific Aim 1) that can be used to engineer any desired additional allele (conditionals, replacements, and point mutations)for each of the 10,000 protein coding regions. 4. Provide a database that allows real-time access to all production data and materials linked to the targetgene as the reference point. 5. Devise a computer program that automates the design of null alleles. 6. Robotically automate the preparation and quality control of BACs and BacVecs. 7. Robotically automate the picking of ES cell clones and their expansion, storage and retrieval. 8. Reduce the unit cost of our automated screening by decreasing reagent use and increase throughput and efficiency by improvements in automated data analysis, targeted clone scoring, and database entry.